JPH0146113B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to a plurality of enzyme activity value analysis methods,
In particular, the present invention relates to an analysis method suitable for efficiently and quickly quantifying enzyme components in a sample containing a large number of substances such as serum.

In recent years, clinical chemistry testing in hospitals has become increasingly mechanized and automated, and especially in the analysis of serum enzymes, the colorimetric method, which measures one point after color development, has changed to the rate method, a method that measures the reaction rate. It is developing. The rate analysis method is the most excellent method for analyzing serum enzymes, but since it measures the reaction rate, it is necessary to determine the amount of change over a predetermined time range, and it requires less time for measurement than single-point measurement. It takes time. In conventional rate analysis, a predetermined amount of sample is dispensed into one reaction container, the reagent is dispensed once or twice to start the enzyme reaction, and the rate of reaction is measured to determine the amount of reagent contained in the sample. One type of enzyme component was being quantified.

On the other hand, put one patient's serum in one container,
When set in an analyzer, several types of components can be analyzed at the same time.
Moreover, there is a demand in the field of clinical testing for an automatic device that can perform multi-item simultaneous analysis by arbitrarily selecting and analyzing one component by the colorimetric method and another by the rate method. Against this background, there is a need for a technology that efficiently combines colorimetric analysis and rate analysis.
Regarding the major differences in measurement between the two analytical methods, the colorimetric method is a method that measures the degree of color development after the chemical reaction that occurs when serum and reagents are mixed. The time required for color development, that is, the reaction time, varies depending on the analysis item and is approximately 15 minutes. However, the measurement time of the reaction solution itself may be 1 second or less. On the other hand, in the rate analysis method, the reaction time can be as short as 2 to 3 minutes, but
The measurement time is long, usually several tens of seconds. Such conventional rate analysis methods only quantify one test enzyme per sample collected in one reaction vessel.

An object of the present invention is to provide a method for analyzing a plurality of enzyme activity values that can efficiently quantitatively analyze a plurality of test enzymes in a sample collected in one reaction vessel.

The present invention includes transporting an array of reaction vessels through first and second reagent addition locations, and disposing a first reagent containing a first substrate and a sample within a particular reaction vessel in the array of reaction vessels. mixing and causing a first enzyme reaction in which the first enzyme to be tested in the sample is involved; and photometrically measuring the reaction solution in which the first enzyme reaction is progressing multiple times to detect the first enzyme to be tested. Quantifying the activity value of the enzyme to be detected, and adding a second reagent containing a second substrate to a reaction vessel containing the reaction solution in which the first enzyme reaction has occurred, and measuring a second test enzyme in the same sample as above. carrying out a second enzymatic reaction involving an enzyme; and measuring the light of the reaction solution in which the second enzyme reaction is progressing multiple times to quantify the activity value of the second enzyme to be tested. Features.

The wavelength for measuring the reaction solution for the first enzyme reaction and the wavelength for measuring the reaction solution for the second enzyme reaction may be different or the same. For example, if the enzymes to be tested are lactate dehydrogenase (LDH) and leucine aminopeptidase (LAP),
The measurement wavelength is different, and the enzymes tested are glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT).
In the case of two items, the measurement wavelengths are the same.

In a preferred embodiment of the present invention, the reaction line is a turntable-shaped reaction disk in which a large number of reaction vessels are arranged concentrically, as a means for efficiently performing multi-item simultaneous analysis using both the colorimetric method and the rate method. One photometer was installed at the sample dispensing position, two reagent dispensing positions, and a position where the absorbance of the reaction solution in the reaction container could be directly measured, and the rotation of the reaction disk was controlled every cycle. It is performed in two phases: stop and rotation. The rotation angle of one cycle is 360 degrees plus 1 pitch, and after adding and mixing the sample and the first reagent, the first reaction is performed for each cycle, for example, one cycle is stopped for 9.5 seconds and rotated for 20.5 seconds, for a total of 30 seconds.
The absorbance of the reaction solution can be measured every 30 seconds, and the reaction rate related to one substance contained in the sample can be determined, thereby allowing the substance to be quantified. A second reagent is then added to the container to cause a relevant reaction in a second substance contained in the same sample, and the absorbance is then measured for multiple cycles to quantify the second substance. do.

Examples of the present invention will be described in detail below.

Embodiment 1 FIG. 1 is a plan view showing a schematic configuration of an apparatus according to an embodiment of the present invention. The reaction disk 1 has a plurality (for example, 40) reaction vessels 2 serving as measurement cells on its circumference, and can freely rotate around a rotation axis 3. The sample table 4 has a plurality of sample containers 5 on its circumference and can freely rotate around a rotation axis 6. Transfer of the sample is performed using a pipette 7 and sampling probe 8, and dispensing of reagents is performed using dispensers 9 and 10.

When performing multi-item simultaneous analysis here, reagent dispensers (not shown) are installed for the number of necessary reagents. The spectrometer 11 is a multi-wavelength simultaneous photometry type having a plurality of detectors, and is opposed to a light source lamp 12, so that when the reaction disk 1 is in a rotating state, the row of reaction vessels 2 passes through the light beam 13 from the light source lamp 12. It is structured as follows. The light beam 13 is arranged so as to pass through the center of, for example, the 31st reaction vessel 2, counting clockwise from the discharge position 25, when the reaction disk 1 is in a stopped state. A drain pipe 26 and a cleaning liquid discharge pipe 27 are provided between the position of the light beam 13 and the discharge position 25.
are arranged and connected to a drainage device 28 and a cleaning device 29, respectively.

FIG. 2 is a block diagram showing the electrical system of the embodiment of FIG. 1. The entire electrical system consists of a multiplexer 14, a logarithmic conversion amplifier 15, and an A/D converter 1.
6, central processing unit 17, read-only storage device 18,
It consists of a read/write storage device 19, a printer 20, an operation panel 21, and a mechanism drive circuit 22, which are connected by a bus line 23.

The operating principle of the embodiment shown in FIG. 1 will be explained below with reference to the drawings. When a sample container 5 containing a sample to be measured, for example, serum, is supplied to the sampling position 30, the tip of the probe 8 of the pipette 7 touches the sample container 5.
A certain amount of serum is aspirated and retained within the probe 8. Thereafter, the probe 8 moves to the discharge position 25 of the reaction disk 1, and
The serum held in the probe 8 is discharged into the reaction container 2, which is being transferred to the reaction vessel 2. When the above-mentioned sampling operation is completed, the reaction disk 1 starts to rotate intermittently in the clockwise direction, and a number of reaction vessels 2 which are one more than the total number of reaction vessels 2, for example 41
Each reaction container 2 rotates by an angle necessary for passing through the discharge position 25, that is, 369 degrees, and then stops.

As the reaction disk 1 rotates, the reaction container 2 containing the sample sampled in the sampling operation comes to a position one reaction container pitch or 9 degrees clockwise from the discharge position 25 and stops. There will be. During the rotation of the reaction disk 1, all reaction vessels 2 on the reaction disk 1 pass through the light beam 13. Therefore, when each reaction container 2 passes through the light beam 13, light absorption is measured by the spectrometer 11, and the output of the spectrometer 11 is selected by the multiplexer 14 as a signal of the currently required measurement wavelength, and then sent to the A/D converter. 16 into the central processing unit 17 and stored in the read/write storage device 19.

If the time period during which the reaction disk 1 is rotated and stopped is, for example, 30 seconds, the above operation is repeated with 30 seconds as one cycle. As the cycle progresses, the position of the sampled sample to be measured advances clockwise by one pitch of the reaction container when the reaction disk 1 is stopped. For example, when the reaction disk 1 is in a stopped state, the pipes of the dispenser 9 and the dispenser 10 are installed above the 1st and 16th reaction vessels 2 in the clockwise direction from the discharge position 25, respectively. Regarding the sample to be measured, the reaction of the first group is started by the first reagent added to the discharge position 1, and then
In the 15th cycle, the second reagent is added by the dispenser 10 to start the second reaction.

As the cycle progresses further, the position of the reaction vessel 2 with the reaction disk 1 stopped is the luminous flux 1.
The sample in the reaction container 2 which is between the light beam 13 and the discharge position 25 beyond 3 is the sample whose measurement has been completed, and is suctioned and drained by the liquid drain device 28 through the liquid drain pipe 26. The cleaning device 29 also passes through the cleaning liquid discharge pipe 27.
A cleaning liquid (usually distilled water) is discharged. When the reaction disk 1 is next stopped, the cleaning liquid in the reaction vessel 2 is drained for the last time in the same manner as described above, and the cycle is further advanced to be used again as the reaction vessel 2 from the discharge position 25.

The above operations are controlled by the central processing unit 17 through the mechanism drive circuit 22 in accordance with the program in the read-only storage device 18 . The operation panel 21 is used for operations such as inputting measurement conditions, starting measurement, and stopping measurement. With the above operation, the stopping time of reaction disk 1 in one cycle is 9.5
Assuming that the rotation time is 20.5 seconds, when focusing on a specific sample, the reaction process of that sample is measured 31 times every 29.5 seconds, and a total of 15 minutes and 15 seconds of measurement data is stored in the read/write storage device 19. ing. The central processing unit 17 operates according to the program in the read-only storage device 18, extracts necessary data from the 31 measurement data in the read/write storage device 19 according to the scheduled program, and performs processing such as concentration calculation. It is then output to the printer 20.

Here, lactate dehydrogenase (LDH) and leucine aminopeptidase (LAP) are specific examples of the present invention.
A case will be described in which the two-component analysis method is applied to the above-mentioned apparatus. As an example of a suitable reagent composition in this case, a solution having the following composition can be used.

First reagent pyruvic acid 0.6m mole/Phosphate buffer (PH7.5) 50m mole/NADH 0.18m mole/Second reagent L-leucine-p-nitroanilide
3.2m mole/phosphate buffer (PH7.5) 400m mole/The measurement conditions of the device are as follows.

Sample amount 20μ First reagent amount 500μ Second reagent amount 250μ Reaction temperature 25℃ Measurement wavelength 1 340nm/376nm Measurement wavelength 2 405nm/505nm Set the above first reagent and second reagent in the dispensers 9 and 10, respectively. When a sample is set on the sample table 4 and a command to start analysis is given from the operation panel, the apparatus operates according to the operating principle described above. If we now track the reaction inside the reaction container 2, the reaction of the following equation (1) will proceed from the time the sample and the first reagent are mixed. LDH, the first enzyme to be tested in the sample
acts on the first substrate, pyruvate, and converts the coenzyme NADH to NAD depending on the LDH content.

Virupic acid + NADHLDH ---→ Lactic acid + NAD... (1) When the second reagent is subsequently added 7.5 minutes later, the reaction of the following equation (2) starts in parallel to the above equation (1).
In the reaction solution where the reaction of formula (1) is progressing, LAP, the second test enzyme in the sample, acts on the second substrate, L-leucine-p-nitroanilide, and Produces aniline.

L-leucine-p-nitroanilide + H ₂ O LAP ---→ L-leucine + p-nitroaniline
…(2) Here, the reaction rate of equation (1) is NADH at 340 nm/
It can be measured by tracking the absorbance using two-wavelength photometry at 376 nm, and this rate is proportional to the amount of LDH activity. Furthermore, the reaction rate of formula (2) can be measured by tracking the absorbance of p-nitroaniline with two-wavelength photometry at 405 nm/505 nm, and the reaction rate determined by this method is proportional to the amount of LAP activity. It is.

As shown in Figure 3, in the case of these two combinations, the measurement wavelength was changed from 340nm/376nm for reaction measurement using the first reagent at the time B when the second reagent was added.
When switching to 405nm/505nm, only the reaction of equation (2) can be measured because there is no component that absorbs at that wavelength among the reaction components of equation (1). In FIG. 3, A is the point of addition of the first reagent. between point A and point B
LDH reaction takes place, and after point B, LAP reaction takes place.

Example 2 Next, a specific example of the present invention will be described with respect to an example of simultaneous two-component analysis of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). As an example of a suitable reagent composition in this case, a solution having the following composition can be used.

The first reagent has the following composition.

α-ketoglutaric acid 18m mole/ L-aspartic acid 200m mole/ NADH 0.18m mole/ MDH 0.6u/ml LDH 1.2u/ml Phosphate buffer (PH7.4) 80m mole/ The second reagent has the following composition.

L-alanine 6.4m mole/ Phosphate buffer (PH7.4) 80m mole/ The measurement conditions of the device are as follows.

Sample amount 20μ First reagent amount 350μ Second reagent amount 50μ Reaction temperature 25℃ Measurement wavelength 340nm/376nm When applied to the apparatus described in Example 1, the reactions of equations (3) and (4) occur after the addition of the first reagent. proceed.

L-aspartic acid + α-ketoglutaric acid GOD ---→ Glutamic acid + Oxaloacetic acid...(3) Oxaloacetic acid + NADH MDH ---→ Malic acid + NAD...(4) Subsequently, when the second reagent is added, equations (3) and (4) are obtained. In parallel with the reaction, the reactions of equations (5) and (1)' proceed.

L-alanine + α-ketoglutaric acid GPT ---→ Glutamic acid + Pyruvate...(5) Pyruvate + NADHLDH ---→ Lactic acid + NAD...(1)' In formula (3), L-aspartic acid, the first substrate, is α - In the presence of ketoglutaric acid, oxaloacetate is produced by the action of GOT, which is the first test enzyme in the specimen, and the produced oxaloacetate participates in the reaction of formula (4). That is, in this example, the first enzyme reaction involving the first test enzyme is
This reaction includes equations (3) and (4). In addition, in formula (5), L-alanine, which is the second substrate, generates pyruvate by the action of GPT, which is the second test enzyme in the sample, in the presence of α-ketoglutaric acid, and this production The resulting pyruvic acid participates in the reaction of equation (1)′. That is, the second enzymatic reaction involving the second test enzyme is a reaction containing formulas (5) and (1)'. Note that LDH in formula (1)' is contained in the first reagent.

The reaction rate in equation (3) is proportional to the GOT concentration in the sample,
It is also proportional to the rate of decrease of NADH in formula (4) conjugated to formula (3), and the rate of decrease in NADH can be measured by absorbance at 340 nm/376 nm. Furthermore, the reaction rate in equation (5) is proportional to the GPT concentration in the sample, and is also proportional to the rate of decrease in NADH in equation (1)', so it can be measured by absorbance at 340 nm/376 nm. That is, as shown in Figure 4, 15 cycles of 7.5 minutes after the first reagent addition.
When calculating the _amount of change in absorbance _per minute _{x 1} from the absorbance data _of . Here, V ₁ : Total reaction solution volume (V = 370 μ) ε: Molecular extinction coefficient (ε = 4.20) d: Optical path length (d = 1 cm) v: Sample amount (v = 20 μ) Y ₁ in equation (6) is _Y1 = _x1 ×370×1000/4.20×1×20= _x1 ×4405…(7) Next, the amount of change in absorbance per minute x ₂ determined from the 15 pieces of absorbance data after addition of the second reagent is proportional to the sum of GOT and GPT (Y ₂ ). In other words, Y ₂ =x ₂ ×V ₂ ×1000/ε×d×v (8), and since V ₂ =420μ, Y ₂ =x ₂ ×420×1000/4.20×1×20=x ₂ × 5000...(9) Therefore, the activity amount Y ₃ of GPT is determined as Y ₃ =Y ₂ -Y ₁ . In FIG. 4, A indicates the time point when the first reagent is added, and B indicates the time point when the second reagent is added.

According to the embodiments described above, there is no waste of specimens, a large number of analyzes can be processed, and the operation is simple and components can be analyzed at one time, so that it can be applied as an efficient multi-item simultaneous analysis system.

As explained above, according to the present invention, a plurality of reactions are caused in a stepwise manner with a time lag in one reaction vessel by one sample sampling, and the rate of change of the reaction solution is measured each time. So,
The number of reaction vessels can be reduced compared to the conventional method, which requires the same number of reaction vessels as the number of analysis items, and
Even though a plurality of test enzymes can be measured in one reaction container, the sample sampling into the reaction container only needs to be done once, so the consumption of valuable samples can be reduced.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of a schematic configuration of an automatic analyzer to which the present invention is applied, FIG. 2 is a block diagram showing the electrical system in the automatic analyzer of FIG. 1, and FIG.
A diagram showing the reaction process of simultaneous analysis of LDH and LAP,
FIG. 4 is a diagram showing the reaction process of simultaneous analysis of GOT and GPT. 1... Reaction disk, 2... Reaction container, 5... Sample container, 7... Pipettor, 9, 10... Reagent dispenser, 11
...spectroscope.

Claims (1)

[Claims] 1. In an analysis method in which the activity value of an enzyme in a sample is determined by mixing a sample and a substrate for enzyme reaction and measuring the rate of decrease in substrate or rate of increase in product, transporting the sample through a sample addition location, mixing the sample with a first reagent containing a first substrate capable of being modified by the first test enzyme in a particular reaction vessel in the array of reaction vessels; and causing a first enzyme reaction involving the first test enzyme in the sample to occur; the first reagent to be added to the reaction vessel at the first reagent addition position; Quantifying the activity value of the first test enzyme by photometrically measuring the reaction solution in which the first enzyme reaction is progressing a plurality of times; , a second substrate that can be modified by a second test enzyme.
Adding a reagent to cause a second enzyme reaction involving the second test enzyme in the sample, and adding the second reagent to the reaction vessel at the second reagent addition position. , and the reaction solution in which the second enzyme reaction is progressing is photometered multiple times to determine the second enzyme reaction.
A plurality of enzyme activity value analysis methods characterized by quantifying the activity value of a test enzyme.